Pyrido[2,3-d]pyrimidine and pyrimido[4,5-d]pyrimidine nucleosides

ABSTRACT

A purine nucleoside analog includes a pyrido[2,3-d]pyrimidine or a pyrimido[4,5-d]pyrimidine and further has a sugar moiety that is optionally modified at the C2′, C3′, C4′ and/or C5′ position. Particularly contemplated compounds also include prodrug forms of the purine nucleoside analogs, and both purine nucleoside analogs and the corresponding prodrugs are employed in the reduction of growth of neoplastic cells.

This application claims the benefit of U.S. provisional application No.60/216,418, filed Jul. 6, 2000, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The field of the invention is nucleoside analogs.

BACKGROUND OF THE INVENTION

Nucleoside analogs have long been used as antimetabolites for treatmentof cancers and viral infections. After entry into the cell, manynucleoside analogs are phosphorylated by nucleoside salvage pathways ina conversion to the corresponding monophosphate by nucleoside kinases,and the monophosphates are subsequently phosphorylated by a kinase tothe di-, and triphosphates. Once a nucleoside analog is converted to itstriphosphate inside the cell, it can serve as a substrate of DNA or RNApolymerases and can be incorporated into DNA or RNA. Incorporation ofcertain unnatural nucleoside analogs into nucleic acid replicates ortranscripts can interrupt gene expression by early chain termination orloss of function of the modified nucleic acids. In addition, certainnucleoside analogs are very potent inhibitors of DNA and RNApolymerases, which can significantly reduce the rate at which thenatural nucleoside can be incorporated.

Moreover, nucleoside analogs can also interfere with a cell in a wayother then DNA and/or RNA synthesis. For example, some nucleosideanalogs may induce apoptosis of cancer cells, or inhibit certain enzymesother than polymerases. In yet further alternative biological effects,some nucleoside analogs are known to modulate the immune system. Typicalexamples for biological effects of nucleoside analogs includethymidylate synthase inhibition by 5-fluorouridine, or adenosinedeaminase inhibition by 2-chloroadenosine. Further examples includeinhibition of S-adenosylhomocysteine hydrolasene by planocin A.

Unfortunately, however, most of the known nucleoside analogs thatinhibit tumor growth or viral infections also imply a threat to thenormal mammalian cells, primarily because such analogs lack adequateselectivity between normal cells and viral or tumor cells. Therefore,there is still a need to provide methods and compositions for nucleosideanalogs with improved specificity and reduced toxicity.

SUMMARY OF THE INVENTION

The present invention provides novel nucleosides having modifications onthe nucleoside base and/or the sugar moiety, which may significantlyincrease selectivity in cytotoxicity to neoplastic cells and/or reducetoxicity of the nucleoside analogs to normal cells. Particularlycontemplated nucleosides include a pyrido[2,3-d]pyrimidine, apyrimido[4,5-d]pyrimidine, and derivatives thereof. The sugar moietiesof contemplated compounds may further include modifications on the C₂,C₃, C₄, and/or C₅ position.

More specifically, the present invention provides nucleosides having astructure according to formula (I):

wherein A is O, S, CH₂; R₂, R₂′, R₃, and R₃′ are independently selectedfrom H, F, OH, NH₂, CN, N₃, CONH₂, and R, where R is lower alkyl, loweralkenyl, lower alkynyl, or lower acyl, and optionally containing atleast one of a heteroatom and a functional group; or R₂ and R₂′together, or R₃ and R₃′ together are selected from ═CH₂, ═CHR″, ═CR″₂,═NR″, where R″ is H, F, OH, CN, N₃, CONH₂, lower alkyl, lower alkenyl,lower alkynyl, or lower acyl; R₄ and R₅′ are independently selected fromH, lower alkyl, lower alkenyl, lower alkynyl, or aralkyl, and optionallycontaining at least one of a heteroatom and a functional group; R₅ is H,OH, OP(O)(OH)₂, P(O)(OH)₂, OP(O)(OR′″)₂, or P(O)(OR′″)₂, where R′″ is amasking group; and B is selected from the group of heterocyclic radicalsconsisting of formula (II), (III), (IV) and (V)

wherein X is H, NH₂ or OH; Y is H, NH₂, or halogen; Z₁ in formula (II)and (IV), and Z₃ in formula (III) and (V) is O, S, NR″, CHM, or CM₂; Z₁in formula (III) and (V), and Z₃ in formula (II) and (IV) is N, CH, orCM; Z₂ is N, CH, or CM; where M is F, Cl, Br, OH, SH, NH₂, CN, COOR″,C(═NH)NH₂, lower alkyl, lower alkenyl, lower alkynyl, aralkyl, or aryl.

In an especially preferred aspect, the nucleoside analog has a structureaccording to formula (VI):

In further contemplated aspects of the inventive subject matter, thenucleoside analogs may be modified to form the respective prodrugs, andparticularly contemplated modifications include phosphorylation oraddition of a phosphonate group at the C₅ position of the sugar moiety,modifications on the hydroxyl groups on the sugar moiety, andmodifications on the amino group of the nucleobase. It is especiallypreferred that such modifications can be cleaved from contemplatedcompounds in a target compartment, target cell, or target organ.

In yet another aspect of the inventive subject matter, a method ofinhibiting growth of a neoplastic cell includes a step in whichcontemplated compounds according to formula (I) are administered to asystem, preferably to a mammal, and more preferably to a human.Especially contemplated neoplastic cells include colon cancer cells,breast cancer cells, melanoma cells, glioma cells, and prostate cancercells. It is further contemplated that the growth inhibition comprisesinhibition of RNA polymerase I, RNA polymerase II, and/or RNA polymeraseIII, and/or induction of apoptosis which may be triggered at least inpart by MEK-phosphorylation.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph depicting comparative cytotoxicity of an exemplarycontemplated compound towards various cancer cells.

FIG. 2 is a graph depicting comparative inhibition of proliferation ofvarious cancer cells by exemplary contemplated compounds.

FIG. 3 is a graph depicting comparative anti-clonogenic activity ofexemplary contemplated compounds in various cancer cells.

FIGS. 4A and 4B are graphs depicting induction of apoptosis in variouscancer cells by exemplary contemplated compounds.

FIG. 5 is a graph depicting inhibition of RNA synthesis in K562 cells byexemplary contemplated compounds.

FIG. 6 is a graph depicting inhibition of RNA Polymerase I & III byexemplary contemplated compounds.

FIG. 7 is a graph depicting inhibition of RNA Polymerase II by exemplarycontemplated compounds.

FIG. 8 is an autoradiograph depicting MEK-ERK phosphorylation byexemplary contemplated compounds.

DETAILED DESCRIPTION OF THE INVENTION

Contemplated Compounds

It is generally contemplated that compounds according to the inventivesubject matter have a structure according to formula (I):

wherein A is O, S, CH₂; R₂, R₂′, R₃, and R₃′ are independently selectedfrom H, F, OH, NH₂, CN, N₃, CONH₂, and R, where R is lower alkyl, loweralkenyl, lower alkynyl, or lower acyl, and optionally containing atleast one of a heteroatom and a functional group; or R₂ and R₂′together, or R₃ and R₃′ together are selected from ═CH₂, ═CHR″, ═CR″₂,═NR″, where R″ is H, F, OH, CN, N₃, CONH₂, lower alkyl, lower alkenyl,lower alkynyl, or lower acyl; R₄ and R₅′ are independently selected fromH, lower alkyl, lower alkenyl, lower alkynyl, or aralkyl, and optionallycontaining at least one of a heteroatom and a functional group; R₅ is H,OH, OP(O)(OH)₂, P(O)(OH)₂, OP(O)(OR′″)₂, or P(O)(OR′″)₂, where R′″ is amasking group; and B is selected from the group of heterocyclic radicalsconsisting of formula (II), (III), (IV), and (V)

wherein X is H, NH₂ or OH; Y is H, NH₂, or halogen; Z₁ in formula (II)and (IV), and Z₃ in formula (III) and (V) is O, S, NR″, CHM, or CM₂; Z₁in formula (III) and (V), and Z₃ in formula (II) and (IV) is N, CH, orCM; Z₂ is N, CH, or CM; where M is F, Cl, Br, OH, SH, NH₂, CN, COOR″,C(═NH)NH₂, lower alkyl, lower alkenyl, lower alkynyl, aralkyl, or aryl.

In a preferred aspect of the inventive subject matter, contemplatedcompounds have a heterocyclic radical according to formula (II), A isoxygen in the sugar moiety of the nucleoside analog, and it is even morepreferred that in such nucleoside analogs X is NH₂, Z₁ is O, and Z₂ andZ₃ are CH. While not limiting the inventive subject matter, it is stillfurther preferred that R₄ and R₅′ are hydrogen, and R₅ is OH in thesugar moiety.

In further particularly preferred aspects of the inventive subjectmatter, contemplated nucleoside analogs have a structure according toformula (VI):

With respect to the stereochemical configuration of contemplatedcompounds, it should be appreciated that the sugar need not berestricted to the D-configuration, but may also be in theL-configuration. Similarly, it is contemplated that suitable moleculesmay include one or more chiral centers, which may be enantiomeric pure(i.e., in R-, or S-configuration) or in a racemic mixture (i e., in R-and S-configuration). Likewise, where substituents (e.g., base or OHgroups) may exhibit an orientation in the α- or β-position, bothpositions are contemplated.

Furthermore, it should be appreciated that contemplated compounds may bemodified to their corresponding prodrug. The term “prodrug” as usedherein refers to any modification of contemplated compounds that (a)changes the molecular weight of contemplated compounds and/or (b) altersthe bioavailability of contemplated compounds with respect to a targetcell and a non-target cell. For example, a prodrug may be prepared byesterification of a hydroxyl group of contemplated compounds with anorganic acid (thereby changing the molecular weight, but not necessarilychanging the bioavailability to a target cell). On the other hand,contemplated compounds may be converted to a prodrug to include a cyclicphosphonate ester (thereby increasing bioavailability to hepatic cells).

Moreover, it should be especially appreciated that prodrug forms ofcontemplated compounds may be entirely or partially re-converted to thecontemplated compounds in a target organ, target cell, or targetcompartment (or in any non-target environment). Reconversion may includevarious mechanisms and especially contemplated mechanisms are enzymaticconversion, oxidation and/or reduction.

Prodrugs may also be employed to increase the specificity ofcontemplated compounds with respect to a target organ, target cell, ortarget compartment. For example, contemplated compounds my be coupled tocholesterol (or a cholesterol derivative) to increase the concentrationof contemplated compounds within the hepato-biliary circulation.Alternatively, contemplated compounds may be coupled to a compound thathas a corresponding receptor on a target cell, thereby increasing theconcentration of contemplated compounds at or in the target cell. In ayet another example, contemplated compounds may be coupled to a nucleartranslocation signal to increase the concentration of contemplatedcompounds within the nucleus of a cell.

Furthermore, prodrugs may be employed to reduce accumulation ofcontemplated compounds in non-target organs, non-target cells, ornon-target compartments. For example, contemplated compounds may bemodified such that a non-target cell will have a significantly reducedrate of uptake of contemplated compounds when such compounds aremodified to a prodrug. Thus, and especially where contemplated compoundsexhibit cytotoxicity to a non-target cell, prodrugs may be employed toreduce cytotoxicity of contemplated compounds in cells or organs otherthan the target cells or target organs.

In yet further contemplated aspects of the inventive subject matter,suitable compounds may be covalently coupled to pharmacologically activeor inactive moieties. For example, pharmacologically active moietiesinclude antineoplastic drugs such as anti-metabolites (e.g.,Pentostatin™), DNA polymerase inhibitors (e.g., Gemzar™), RNA polymeraseinhibitors (e.g, ECyd™), platinum derivatives (e.g., Paraplatin™),anti-estrogens (e.g., Nolvadex™), Taxanes (e.g., Taxotere™), GnRHanalogs (e.g., Lupron™), DNA polymerase inhibitors (e.g., Gemzar™),topoisomerase inhibitors (e.g., Hycamptin™), biphosphonates (e.g.,Aredia™), somatostatins (e.g., Sandostatin™), interferons (e.g.,IntronA™), nucleoside analogs (e.g., Ribavirin™), and IMPDH-inhibitors(e.g., Tiazofurin™).

Pharmacologically inactive moieties include biological andnon-biological moieties. For example, where target specificity isparticularly desirable, contemplated compounds may be coupled to anantibody, an antibody fragment, or a synthetic antibody (e.g, scFv). Ina further example, contemplated compounds may be coupled to a chelator(e.g., that binds a radionucleid). Alternatively, where prolongation ofserum half-life or reduced immunogenicity is particularly preferred,contemplated compounds may be coupled to inert or biodegradable polymers(e.g., dextran, polyethylene glycol, etc.).

With respect to the mode of coupling contemplated compounds to othermoieties, all known methods of coupling are considered appropriate andespecially include covalent coupling (with or without a separate linkermolecule), hydrogen bonding, and hydrophobic/hydrophilic interactions.

In still further aspects of the inventive subject matter, contemplatedcompounds may also be in the form of their respective salts, wherein thesalt may be a salt of an organic or inorganic acid or base (e.g.,actetate or morpholino salt, HCl salt). There are numerouspharmacologically acceptable salts known in the art, and all of theknown salts are considered suitable for use in conjunction with theteachings presented herein.

Synthesis of Contemplated Compounds

Synthesis of Modified Ribofuranoses

The exemplary schemes below show the synthetic routes to some of thecontemplated compounds. Compound 1, prepared according to a publishedprocedure (Jones et al. Methods in Carbohydrate Chemistry (edited byWhistler and Moffat), vol. VI, pp315–322, Academic Press, New York,(1972)), was treated with a variety of nucleophiles such as Grignardreagents to give 2, which was benzoylated or acetylated and subsequentlytreated with trifluoroacetic acid to give compound 4. Benzoylation andthe subsequent treatment with acetic anhydride/acetic acid in thepresence of sulfuric acid gave compound 6, which was used forcondensation with pyrido[2,3-d]pyrimidine or pyrimido[4,5-d]pyrimidinebases.

Compound 2a (5(R)-C-methyl derivative) was converted to the sulfonate 7,which was subjected to nucleophilic substitution to give theconfigurationally inverted compound 8. Deprotection of theisopropylidene and the subsequent acetylation gave the tetraacetate 9.

Compound 1 was treated with formaldehyde in aqueous sodium hydroxide togive 4′-hydroxymethyl derivative 10, which was selectively protected toafford compound 11.

The subsequent protection with DMT and removal of TBS gave compound 13,which can be converted to a variety of substituents. The 4-C-substitutedderivatives subjected to the similar transformations as 5-C-substitutedribofuranoses can be converted to compound 17, which is used forcondensation with nucleoside bases.

Compound 13 was converted to 18, which was subjected to Wittig reactionto give 19. Hydrogenation of 19 over palladium afforded 20. Compound 13was converted to the 4-C-phenoxythiocarbonyloxymethyl derivative 21,which was reacted with tris(trimethylsilyl)silane (9.0 mL, 29 mmol) andthen with 1,1′-azobis(cyclohexanecarbonitrile) to give 22.

Where contemplated compounds include a sugar moiety that is not modifiedat the C₄′ or C₅′ position (e.g., L-ribofuranose,2′-hydroxy-2′-ethynyl-L-ribofuranose), condensation of the correspondingprotected sugar moiety (in D- or L-configuration) is carried outfollowing procedures well known in the art.

Synthesis of Modified Pyridopyrimidine Nucleosides

The 5′-substituted nucleoside analogs are prepared from the condensationof the silylated pyrido[2,3-d]pyrimidine bases and the properlyprotected, modified ribofuranoses. The following scheme shows synthesisof 4-amino-5-oxo-pyrido[2,3-d]pyrimidine 25. Compound 23, preparedaccording to a reported procedure (Archive der Pharmazie 1985, 318,481–486), was refluxed with chlorotrimethylsilane and sodium iodide inacetonitrile to give 24. Reaction of 24 with formamidine acetate underreflux afforded compound 25.

The following schemes show the condensation of thepyrido[2,3-d]pyrimidine 25 and the modified ribofuranoses 6 and 17.Compound 25 was treated with (bis(trimethylsily)acetamide to give thesilylated pyridopyrimidine, which was reacted with 6 or 17 to give 26 or28. Removal of benzoyl afforded the pyrido[2,3-d]pyrimidine nucleosides27 and 29, respectively.

Other sugar-modified pyridopyrimidine or pyrimidopyrimidine nucleosidescan be prepared either through condensation of nucleoside bases andmodified sugars or through modifications of the nucleosides. Forexample, 4-amino-5-oxo-pyrido[2,3-d]pyrimidine riboside (27 or 29 R═H)was converted to 2′-deoxy derivative 30 by a similar procedure describedfor 2′-deoxyadenosine, and 4-amino-5-oxo-pyrido[2,3-d]pyrimidinexyloside 34 was prepared by condensation.

4-Amino-5-oxopyrido[2,3-d]pyrimidine was condensed with a variety of1-O-acetylated pentose sugars via Vorbrüggen reactions. Furtherderivatizations of the nucleosides provided an additional group ofpyrido[2,3-d]pyrimidine nucleosides.

Synthesis of Modified Pyrimidopyrimidine Nucleosides

The 5′-substituted nucleoside analogs are prepared from the condensationof the silylated pyrimido[4,5-d]pyrimidine bases and the properlyprotected, modified ribofuranoses. The synthesis ofpyrimido[4,5-d]pyrimidine nucleosides follows a protocol substantiallysimilar to the synthetic procedure as outlined above.

Depending on the reaction conditions of the condensation reactionsemployed to couple the sugar moiety to the pyrido[2,3-d]pyrimidine orpyrimido[4,5-d]pyrimidine base, the corresponding nucleoside analogs maybe connected at the N₁ or N₈ atom of the base. In any case, theglycoslyation site of the base was established by X-ray crystalstructure.

Uses of Contemplated Compounds

It should generally be recognized that the contemplated compounds may beemployed in any treatment or therapy of a system that positivelyresponds to administration of contemplated compounds. However, it isparticularly preferred that the contemplated compounds may be employedin antineoplastic treatments and antiviral treatments (as a directantiviral compound and/or as an indirect antiviral compound), and intreatments to modulate the immune system.

Antineoplastic Treatments

It is generally contemplated that compounds according to the inventivesubject matter may be employed as antineoplastic agents that directly orindirectly inhibit growth, invasiveness, and/or spread of a neoplasticcell or cell population. It is particularly contemplated that a methodof treating a neoplastic disease in a patient comprises a step in whichthe contemplated compounds are administered to the patient at a dosageeffective to inhibit growth of a neoplastic cell, and an especiallypreferred compound is the compound according to formula (VI, supra).Contemplated dosages are in the range between 0.01–100 mg/kg, and morepreferably between 5–50 mg/kg. However, alternative dosages, routes,schedules and formulations are also contemplated, and suitablealternative administrations are described below. While the use ofcontemplated compounds is not restricted to a particular neoplastic cellor neoplastic disease, especially contemplated neoplastic cells includecolon cancer cells, breast cancer cells, melanoma cells, glioma cells,and prostate cancer cells.

Antiviral Treatments

It is generally contemplated that compounds according to the inventivesubject matter may be employed as a direct and/or indirect antiviralagent in a viral infection. It is particularly contemplated that amethod of treating a viral infection in a patient comprises a step inwhich the contemplated compounds are administered to the patient at adosage effective to inhibit viral propagation (i.e., a process involvinga host cell in which one or more than one virus causes the host cell toproduce one or more copies of the virus, wherein the term “to produce”refers to nucleotide synthesis, protein processing, and proteinassembly), and wherein the composition comprises at least one of thecontemplated compounds. Contemplated dosages are in the range of between0.1–100 mg/kg, and more preferably between 5–50 mg/kg. However,alternative dosages, routes, schedules and formulations are alsocontemplated, and suitable alternative administrations are describedbelow. While the use of contemplated compounds is not restricted to aparticular virus in a particular viral infection, especiallycontemplated viral infections are an HIV infection, an HCV infection, anHBV infection, an RSV infection, an influenza virus infection, and aparainfluenza virus infection.

Immunomodulation

It is generally contemplated that compounds according to the inventivesubject matter may be employed as immunomodulatory compounds, and it isparticularly contemplated that such compounds may be employed tomodulate the balance between a Type 1 response and a Type 2 response ofan immunocompetent cell (e.g., T-cell) towards a challenge. Morespecifically, it is contemplated that the compounds according to theinventive subject matter may increase the Type 1 response relative to aType 2 response (either by increasing the Type 1 response or bydecreasing the Type 2 response), however, it is also contemplated thatthe compounds of the inventive subject matter may increase the Type 2response relative to a Type 1 response (either by increasing the Type 2response or by decreasing the Type 1 response). In still farthercontemplated uses of the compounds according to the inventive subjectmatter, it should be appreciated that contemplated compounds may also beemployed as immunosuppressive agents at a concentration effective tosuppress both Type 1 and Type 2 responses.

Administration of Contemplated Compounds

With respect to administration of contemplated compounds, it should beappreciated that the compounds may be administered under any appropriateprotocol in any appropriate pharmaceutical formulation. It is generallypreferred, however, that contemplated compounds are orally administered.In further aspects of the inventive subject matter, it should beappreciated that various alternative administrations are also suitable,and it should still further be recognized that a particularadministration will generally depend on chemical stability,bioavailability, dosage, formulation, and/or desiredpharmacokinetic/pharmacodynamic properties of contemplated compounds.Thus, appropriate administrations will include oral administration(e.g., tablet, syrup, etc.), topical delivery (e.g., ointment, spray,cream, etc.), parenteral systemic delivery (e.g., inhalation), anddirect or indirect delivery to the blood stream (e.g., i.v. or i.m.injection, etc.).

Consequently, the formulation of contemplated compounds may varyconsiderably. For example, where the drug or drug composition exhibitssufficient stability to pass through the gastro-intestinal systemwithout undesired chemical or enzymatic modification, oral formulationsmay include syrup, tablets, gel caps, powder, etc. On the other hand,where absorption or passage of contemplated compounds through thegastrointestinal tract into the blood stream is problematic, suitableformulations especially include injectable solutions or suspensions(e.g., physiological saline solution buffered to a pH of about 7.2 to7.5).

With respect to the dosage of contemplated compounds, it should beappreciated that various dosages are suitable, and contemplated dosagestypically are in the range of 0.1 mg/kg to several 100 mg/kg, and evenmore. For example, where contemplated compounds are excreted ormetabolized at a relatively low rate, or where long-term treatment isdesired, dosages will typically be in the range between 0.5 mg–10 mg/kg.On the other hand, where bioavailability of contemplated drugs isrelatively low, or where metabolic conversion is relatively fast,dosages will typically be in the range between 10 mg/kg–100 mg/kg.

With respect to the dosage of contemplated compounds, it should furtherbe appreciated that at least some of the compounds according to theinventive subject matter may be phosphorylated in vivo. Consequently,and especially where immediate bioavailability is desired, dosages maybe reduced where contemplated compounds are administered in aphosphorylated form.

The schedule of administration may vary considerably, and contemplatedschedules include a single dose over the entire course of treatment,multiple single daily doses over the entire course of treatment,multiple daily doses, and permanent dosing (e.g., permanent infusion,implanted osmotic pump, etc.) for at least part of the course oftreatment. While it is generally preferred that suitable schedulessustain constant delivery of contemplated compounds, burst delivery(i.e., at least one administration at a first dose followed by at leastone more administration at a dose lower than the first dose) is alsoappropriate. With respect to the duration of treatment, it iscontemplated that appropriate durations may vary between a singleadministration and several days, several weeks, several years, and evenlonger. For example, where contemplated compounds are employed in a cellculture, a single administration, or relatively short administration maybe sufficient. On the other hand, where contemplated compounds areadministered to treat an acute phase of a disease, appropriate treatmentduration may be in the range between several days and several weeks.Similarly, where chronic diseases are treated by administration ofcontemplated compounds, extended administration over one or more yearsmay be suitable.

In still further alternative aspects of the inventive subject matter,contemplated compounds may be combined with additional pharmaceuticallyactive substances to assist in the treatment of various diseases, andparticularly neoplastic diseases. Additional pharmaceutically activesubstances may be administered separately or together, and whenadministered separately, administration may occur simultaneously orseparately in any order. Especially contemplated additionalpharmaceutically active substances include drugs commonly used aschemotherapy for treatment of cancer and immune modulator substances.For example, chemotherapeutic agents include anti-metabolites (e.g.,Pentostatin™), DNA polymerase inhibitors (e.g, Gemzar™), RNA polymeraseinhibitors (e.g., ECyd™), platinum derivatives (e.g., Paraplatin™),anti-estrogens (e.g., Nolvadex™), Taxanes (e.g., Taxotere™), GnRHanalogs (e.g., Lupron™), DNA polymerase inhibitors (e.g., Gemzar™),topoisomerase inhibitors (e.g., Hycamptin™), biphosphonates (e.g.,Aredia™), somatostatins (e.g., Sandostatin™), nucleoside analogs (e.g.,Ribavirin™), and IMPDH-inhibitors (e.g., Tiazofurin™). Contemplatedimmunomodulatory substances include cytokines (e.g., interferon α and γ,IL2, IL4, IL6, IL8, IL10, and IL12), cytokinins (e.g., kinetin), andchemokines (e.g., MIP-1).

EXAMPLES

The following examples provide exemplary synthesis, in vitro/in vivoexperiment, and are intended to illustrate but not to limit theinvention.

Synthesis

Preparation of2,3-O-isopropylidene-5(R,S)-C-ethynyl-1-O-methyl-β-D-ribofuranose

To a stirred solution of methyl4-C,5-O-didehydro-2,3-O-isopropylidene-β-D-ribofuranoside (Jones et al.Methods in Carbohydrate Chemistry Vol 1, pp 315–322 (1972), 4.00 g,19.78 mmol) in anhydrous THF (20 mL) at −42° C. under argon was addeddropwise ethynylmagnesium bromide (0.5 M in THF, 80 mL, 40 mmol). Uponaddition, the resulting mixture was slowly warmed up to 0° C. (˜90min.). The reaction was quenched by adding ice (50 g)/water (50 mL) andthe mixture was stirred for 30 min. After neutralization with 10% aq.acetic acid, the mixture was extracted with ethyl acetate twice. Thecombined organic layer was dried (Na₂SO₄) and concentrated.Chromatography on silica (ethyl acetate-hexanes 1:4) gave 3.48 g of thetitled compound (R/S ratio 1:1) as a white solid. Similarly, thefollowing compounds were prepared:1-O,5(R)-C-Dimethyl-2,3-O-isopropylidene-β-D-ribofuranose from4-C,5-O-didehydro-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose andmethylmagnesium bromide;2,3-O-Isopropylidene-1-O-methyl-5(R)-C-vinyl-β-D-ribofuranose from4-C,5-O-didehydro-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose andvinylmagnesium bromide;5(R)-C-Allyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose from4-C,5-O-didehydro-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose andallylmagnesium bromide.

Preparation of 5-O-acetyl-1-O,5(S)-C-dimethyl-2,3-O-isopropylidene-β-Dribofuranose

To a stirred solution of1-O,5(R)-C-dimethyl-2,3-O-isopropylidene-β-D-ribofuranose (7.24 g, 33.17mmol) in anhydrous pyridine (50 mL) at 0° C. was added methanesulfonylchloride (3.1 mL, 39.92 mmol). The resulting mixture was stirred at roomtemperature for 1 h, cooled to 0° C., quenched by adding water (1.0 mL),and stirred at room temperature for 30 min. The solvent was evaporatedand the residue was dissolved in ethyl acetate, washed with brine threetimes, dried (Na₂SO₄) and concentrated. Chromatography on silica (30%EtOAc in hexanes) gave 8.62 g of the methylate as a colorless syrup.

A stirred suspension of1-O,5(R)-C-dimethyl-2,3-O-isopropylidene-5-O-methanesulfonyl-β-D-ribofuranose(8.62 g, 29.1 mmol) and NaOAc (anhydrous, 3.5 g, 42.5 mmol) in anhydrousDMF (350 mL) was heated at 125° C. under argon for 4 days. The solventwas evaporated and the residue chromatographed on silica (25% EtOAc inhexanes) to give 4.0 g of the titled compound as a white solid.

Preparation of 5-deoxy-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose

To a stirred solution of2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose (14.2 g, 70.0 mmol) inanhydrous pyridine (250 mL) at 10° C. was added in portions (over 30min) p-toluenesulfonyl chloride (19.1 g, 100 mmol). The resultingmixture was stirred at room temperature for 18 h, cooled to 0° C.,quenched by adding water (5.0 mL), and stirred at room temperature for30 min. The solvent was evaporated. The residue was dissolved in ethylacetate, washed with brine three times, dried (Na₂SO₄) and concentratedto dryness. Chromatography on silica (ethyl acetate-hexanes 1:3) gave24.1 g of the tosylate as a white solid.

To a stirred suspension of LiAlH₄ (4.58 g, 120.5 mmol) in anhydrousdiethyl ether (120 mL) was added the tosylate (13.1 g, 36.55 mmol) indiethyl ether-toluene (2.5:1, 140 mL). The resulting mixture wasrefluxed for 22 h, cooled to room temperature, diluted with ethylacetate (25 mL) quenched by adding water (5.0 mL). The solvent wasevaporated. The residue was dissolved in ethyl acetate, washed withbrine three times, dried (Na₂SO₄) and concentrated to dryness.Chromatography on silica (ethyl acetate-hexanes 1:3) gave 3.58 g of thetitled compound as a colorless liquid.

Preparation of 5(R)-C-allyl-5-O-benzoyl-1-O-methyl-β-D-ribofuranose

To a stirred solution of5(R)-C-allyl-2,3-O-isoproplidene-1-O-methyl-β-D-ribofuranose (4.49 g,18.38 mmol) in anhydrous pyridine (40 mL) at 0° C. was added benzoylchloride (2.7 mL, 23.0 mmol). The resulting mixture was stirred at roomtemperature for 18 h, cooled with ice, quenched by adding water (1 mL),and stirred at room temperature for 30 min. The solvent was evaporatedand the residue was dissolved in ethyl acetate, washed with brine threetimes, dried (Na₂SO₄) and concentrated. Chromatography on silica (12%ethyl acetate in hexanes) gave 6.26 g of5(R)-C-allyl-5-O-benzoyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranoseas a colorless syrup.

A solution of5(R)-C-allyl-5-O-benzoyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose(6.2 g, 17.8 mmol) in TFA-H₂O mixture (9:1) was stirred at 0° C. for 90min and concentrated to dryness at 0° C. The residue was dissolved inmethanol-toluene mixture (20 mL, 1:1) and concentrated to dryness.Chromatography on silica (ethyl acetate-hexanes 1:1) gave 3.70 g of thetitled compound as a white solid. Similarly, the following compoundswere prepared: 5-O-Benzoyl-5(R,S)-C-ethynyl-1-O-methyl-β-D-ribofuanose(R/S ratio: 1:1) from5-O-benzoyl-5(R,S)-C-ethynyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose;5-O-Benzoyl-4-C-benzoyloxymethyl-1-O-methyl-β-D-ribofuranose from5-O-benzoyl-4-C-benzoyloxymethyl-2,3-O-isopropylidene1-O-methyl-β-D-ribofuranose;5-O-Benzoyl-1-O-methyl-5(R)-C-vinyl-β-D-ribofuranose from2,3-O-isopropylidene-1-O-methyl-5(R)-C-vinyl-β-D-ribofuranose.

Preparation of1-O-acetyl-5(R)-C-allyl-2,3,5-tri-O-benzoyl-D-ribofuranose

To a stirred solution of 5(R)-C-allyl-5-O-benzoyl-1-O-methyl-62-D-ribofuranose (3.60 mg, 11.68 mmol) in anhydrous pyridine (80 mL) at0° C. was added benzoyl chloride (3.0 mL, 25.84 mmol). The resultingmixture was stirred at room temperature for 18 h, cooled with ice,quenched by adding water (1 mL), then stirred at room temperature for 30min. The mixture was concentrated, diluted with ethyl acetate, washedwith brine three times, dried (Na₂SO₄) and concentrated to dryness.Chromatography on silica (15% ethyl acetate in hexanes) gave 5.3 g ofthe titled compound as a colorless syrup.

To a stirred solution of5(R)-C-allyl-2,3,5-tri-O-benzolyl-1-O-methyl-β-D-ribofuranose (4.0 g,7.74 mmol) in acetic acid (14 mL) and acetic anhydride (1.75 mL, 18.36mmol) at 0° C. was added concentrated sulfuric acid (200 μL, 3.79 mmolin 4.0 mL of acetic acid). The resulting mixture was stirred at roomtemperature for 20 h, cooled to 0° C., diluted with cold ethyl acetate,washed with water, dilute NaHCO₃ and then brine, dried (Na₂SO₄), andconcentrated. Chromatography on silica (ethyl acetate-hexanes 1:4) gave2.82 g of the titled compound (α/β ratio: 1:2) as a colorless foam.Similarly, the following compounds were prepared:1-O-Acetyl-5(R,S)-C-ethynyl-2,3,5-tri-O-benzolyl-β-D-ribofuranose (R/Sratio: 1:1 and α/β ratio: 1:2) from methyl5(R,S)-C-ethynyl-2,3,5-tri-O-benzolyl-β-D-ribofuranoside;1-O-Acetyl-4-C-benzoyloxymethyl-2,3,5-tri-O-benzoyl-D-ribofuranose (α/βratio: 1:3) from methyl4-C-benzoyloxymethyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside;5(R)-C-Methyl-1,2,3,5-tetra-O-acetyl-β-D-ribofuranose from1-O,5(R)-C-dimethyl-2,3-O-isopropylidene-β-D-ribofuranose;5(S)-C-Methyl-1,2,3,5-tetra-O-acetyl-β-D-ribofuranose from5-O-acetyl-1-O,5(R)-C-dimethyl-2,3-O-isopropylidene-β-D-ribofuranose;5-Deoxy-1,2,3-tri-O-acetyl-β-D-ribofuranose from5-O-acetyl-2,3-O-isopropylidene1-O-methyl-β-D-ribofuranose;1-O-Acetyl-2,3,5-tri-O-benzoyl-5(R)-C-vinyl-β-D-ribofuranose from1-O-methyl-2,3,5-tri-O-benzoyl-5(R)-C-vinyl-β-D-ribofuranose.

Preparation of1-O-methyl-5-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranose

To a stirred solution of4-C,5-O-didehydro-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose 1(20.22 g, 100 mmol) in dioxane (380 mL) at 0° C. was added dropwiseformaldehyde (37% solution, 76 mL) and then 2 M NaOH (188 mL). Theresulting reaction mixture was stirred at room temperature for 20 h,cooled to 0° C., neutralized (10% acetic acid), concentrated (˜50%), andextracted with methylene chloride twice. The combined organic layer wasdried over Na₂SO₄ and concentrated to dryness. Chromatography on silica(4% methanol in chloroform) gave 20.2 g of1-O-methyl-5-O-benzoyl-2,3-O-isopropylidene-4-C-benzoyloxymethyl-β-D-ribofuranoseas a white solid.

A solution of1-O-methyl-5-O-benzoyl-2,3-O-isopropylidene-4-C-benzoyloxy-methyl-β-D-ribofuranose(2.0 g, 4.5 mmoles) in a 9:1 (v/v) mixture of trifluoroacetic acid andwater (11 mL) was stirred at 0° C. for 2 h and evaporated to dryness.The residue was dissolved in methanol and evaporated (3 times), thendissolved in pyridine and evaporated. The residue was subjected tosilica gel chromatography [methanol (0–0.5%) in dichloromethane] to give1.7 g of the titled compound as an oil.

Preparation of1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranose

To a solution of1-O-methyl-5-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranose (1.7 g, 4.2mmoles) in pyridine (14 mL) was added benzoyl chloride (1.2 mL, 10mmoles). The reaction mixture was stirred at 25° C. for 16 h andmethanol (5 mL) was added. The solvents were evaporated and the residuewas dissolved with ethyl acetate (20 mL) and water (10 mL). The organiclayer was dried over sodium sulfate, filtered, and the filtrate wasevaporated to dryness. The residue was subjected to silica gelchromatography [methanol (0–0.5%) in dichloromethane] to give 2.4 g ofPreparation of1-O-methyl-2,3,5-tri-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranose asa white solid.

Sulfuric acid (97%, 75 mL) was added to a solution of1-O-methyl-2,3,5-tri-O-benzoyl-4-C-(benzoyloxymethyl)-β-D-ribofuranose(1.7 g, 2.8 mmoles) in a mixture of acetic acid (6.7 mL) and aceticanhydride (0.67 mL) at 0° C. The reaction mixture was stirred at 25° C.for 15 h and diluted with ethyl acetate (50 mL) and water (10 mL). Thissolution was washed with brine (3 times), with a saturated aqueoussolution of sodium bicarbonate, dried over sodium sulfate, filtered, andthe filtrate was evaporated to dryness. The residue was subjected tosilica gel chromatography [ethanol (0–2%) in dichloromethane] to give1.4 g of the titled compound as a white solid.

Preparation of4-C-(4,4′-dimethoxytrityloxymethyl)-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose

A solution of 4,4′-dimethoxytrityl chloride (6.0 g, 18 mmol) in pyridine(18 mL) was added to a solution of4-C-hydroxymethyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose (3.5g, 15 mmol) in pyridine (60 mL) stirred at 0° C. The reaction mixturewas stirred at room temperature for 18 h, then cooled to 0° C. Methanol(6 mL) was added, and the solvents were evaporated under reducedpressure. Ethyl acetate and brine were added, and the organic extractwas washed with brine, dried over sodium sulfate, filtered andevaporated to dryness. The residue was subjected to silica gelchromatography [ethyl acetate (20–25%) in hexanes] to give 6.2 g of thetitled compound as a white solid.

Preparation of2,3,5-tri-O-benzoyl-1-O-methyl-4-C-methyl-β-D-ribofuranose

A solution of benzoyl chloride (1.5 mL, 13 mmol) in pyridine (6 mL) wasadded to a solution of4-C-(4,4′-dimethoxytrityloxymethyl)-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose(6.2 g, 12 mmol) in pyridine (52 mL) stirred at 0° C. The reactionmixture was stirred at room temperature for 15 h, then cooled to 0° C.Methanol (5 mL) was added, and the solvents were evaporated underreduced pressure. Ethyl acetate and brine were added, and the organicextract was washed with brine, dried over sodium sulfate, filtered andevaporated to dryness. The residue was co-evaporated with toluene anddissolved in a solution of 80% of acetic acid in water (174 mL). Thereaction mixture was stirred at room temperature for 2 hours, then thesolvent was evaporated under reduced pressure. The residue was subjectedto silica gel chromatography [methanol (1–2%) in dichloromethane] toremove most of the impurities, and the white foam obtained was dissolvedin acetonitrile (174 mL). This solvent was stirred at 0° C. and thenN,N-dimethylaminopyridine (4.3 g, 35 mmol) and phenoxythiocarbonylchloride (2.4 mL, 17 mmol) were added. The reaction mixture was stirredat room temperature for 2 h again and the solvent was evaporated. Theresidue was dissolved with dichloromethane and water, and the resultingorganic extract was washed with a 0.5 N solution of hydrochloric acid,then with water and finally with brine. It was dried over sodiumsulfate, filtered, and the filtrate was evaporated under reducedpressure. The residue was dissolved with toluene, andtris(trimethylsilyl)silane (9.0 mL, 29 mmol) and1,1′-azobis(cyclohexanecarbonitrile) (0.71 g, 2.9 mmol) were added. Thereaction mixture was stirred at 100° C. for 15 h, then cooled to roomtemperature and the solvent was evaporated under reduced pressure. Theresidue was subjected to silica gel chromatography [methanol (1–2%) indichloromethane] to remove most of the impurities, and the oil obtainedwas dissolved in a −15° C. solution of trifluoroacetic acid in water(90% v/v, 21 mL). The reaction mixture was stirred at −10° C. for 1 h,then the solvent was evaporated under high vacuum and low temperature.The residue was coevaporated with methanol and subjected to silica gelchromatography [methanol (0–3%) in dichloromethane] to remove most ofthe impurities. The oil obtained was dissolved with pyridine (18 mL) andthe solution was stirred at 0° C. Benzoyl chloride (1.4 mL, 12 mmol) wasadded and the reaction mixture was stirred at room temperature for 16 h,then cooled to 0° C. Methanol was added and the solvents were evaporatedunder reduced pressure. Ethyl acetate, hexanes and brine were added tothe residue, and the resulting organic extract was dried over sodiumsulfate, filtered and the filtrate was evaporated to dryness. Theresidue was subjected to silica gel chromatography [ethyl acetate (25%)in hexanes] to give 1.8 g (32%, 6 steps) of the titled compound as asyrup.

Preparation of1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-methyl-β-D-ribofuranose

Concentrated sulfuric acid (97%, 99 mL) was added to a solution of2,3,5-tri-O-benzoyl-1-O-methyl-4-C-methyl-β-D-ribofuranose (1.8 g, 3.6mmoles) in a mixture of acetic acid (9.0 mL) and acetic anhydride (0.90mL) at 0° C. The reaction mixture was stirred at 25° C. for 16 h anddiluted with ethyl acetate (50 mL) and brine (10 mL). The organicextract was washed with brine (3 times), with a saturated aqueoussolution of sodium bicarbonate, dried over sodium sulfate, filtered, andthe filtrate was evaporated to dryness. The residue was dissolved with amixture of ethyl acetate and hexanes (1:4, v/v) and the b anomer of thetitle compound crystallized instantaneously. The white crystals werefiltered to give 1.1 g (56%) of the title compound in its pure b anomerform. The filtrate was subjected to silica gel chromatography [ethylacetate (20%) in hexanes] to give 0.6 g (32%) of the titled compound asan oil (3:1 mixture of a/b anomers).

Preparation of5-O-(4,4′-dimethoxytrityl-4-C-hydroxymethyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose

A solution of tert-butyldimethylsilyl chloride (3.4 g, 23 mmol) inpyridine (16 mL) was added to a solution of4-C-hydroxymethyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose (4.5g, 19 mmol) in pyridine (80 mL), and stirred at 0° C. The reactionmixture was then stirred at room temperature for 24 h, then cooled to 0°C. Water (5 mL) was added, and the solvent was evaporated under reducedpressure. Ethyl acetate and brine were added, and the organic extractwas washed with brine, dried over sodium sulfate, filtered andevaporated to dryness. The residue was dissolved in pyridine and thesolution was stirred at 0° C. 4,4′-Dimethoxytrityl chloride (8.4 g, 25mmol) was added, and the reaction mixture was stirred at roomtemperature for 16 hours, then cooled to 0° C. Methanol (10 mL) wasadded, and the solvents were evaporated under reduced pressure. Ethylacetate, hexanes and brine were added, and the organic extract waswashed with a 0.5 N solution of hydrochloric acid, then with brine,dried over sodium sulfate, filtered and evaporated to dryness. Theresidue was dissolved in tetrahydrofuran (57 mL), and a solution oftetrabutylammonium fluoride (TBAF, 1 M in tetrahydrofuran, 23 mL) wasadded. After 24 h at room temperature, 0.2 equivalent of TBAF was added,and the mixture was stirred for an additional 36 h. The solvent wasevaporated and the residue was subjected to silica gel chromatography[ethyl acetate (50%) in hexanes] to give 6.6 g (65%, 3 steps) of thetitled compound as a white solid.

Preparation of5-O-(4,4′-dimethoxytrityl)-2,3-O-isopropylidene-1-O-methyl-4-C-vinyl-β-D-ribofuranose

A solution of trifluoroacetic acid (0.49 mL, 6.4 mmol) and pyridine (1.6mL, 19 mmol) in dimethylsulfoxide (11 mL) was added to a solution of5-O-(4,4′-dimethoxytrityl)-4-C-hydroxymethyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose(6.9 g, 13 mmol) and N,N′-dicyclohexylcarbodiimide (6.6 g, 32 mmol) in amixture of toluene (26 mL) and dimethylsulfoxide (66 mL) stirred at 5°C. The reaction mixture was then stirred at room temperature for 8 h,then cooled to 0° C. Ethyl acetate (80 mL) and a solution of oxalic acid(1.8 g, 19 mmol) in methanol (10 mL) were added, and the mixture wasstirred at room temperature for 15 h. The precipitate was filtered andwashed with a 1:1 mixture of hexanes and ethyl acetate. The filtrate waswashed with brine, with a saturated aqueous solution of sodiumbicarbonate, washed with brine again, dried over sodium sulfate,filtered, and the filtrate was evaporated to dryness. The residue wassubjected to silica gel chromatography [ethyl acetate (25%) in hexanes]to give 6.1 g (89%) of5-O-(4,4′-dimethoxytrityl)-4-C-formyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranoseas a white solid.

A solution of sodium pentoxide (2.5 g, 22 mmol) in benzene (34 mL) wasadded to a suspension of methylphosphonium bromide (8.8 g, 25 mmol) inether stirred at room temperature. The mixture was stirred at roomtemperature for 6 h, and a solution of5-O-(4,4′-dimethoxytrityl)-4-C-formyl-2,3-O-isopropylidene-1-O-methyl-β-D-ribofuranose(6.0 g, 11 mmol) in ether (30 mL) was added. The resulting mixture wasstirred at room temperature for 18 h, then cooled to 0° C. Brine (100mL) was added, followed by ethyl acetate (300 mL). The organic extractwas washed with brine, dried over sodium sulfate, filtered and thefiltrate was evaporated to dryness. The residue was subjected to silicagel chromatography [ethyl acetate (25%) in hexanes] to give 5.9 g (99%)of the titled compound as a white solid.

Preparation of 5-O-benzoyl-4-C-ethyl-1-O-methyl-b-D-ribofuranose

Palladium on activated carbon (10% Pd, 50% water, 508 mg) was added to asolution of5-O-(4,4′-dimethoxytrityl)-2,3-O-isopropylidene-1-O-methyl-4-C-vinyl-β-D-ribofuranose(5.0 g, 9.4 mmol) in methanol (254 mL). The flask was shaken under 5 psiof hydrogen during 6 h and the catalyst was filtered and washed withmethanol. The solvent was evaporated, and the residue was coevaporatedwith pyridine. It was then dissolved in pyridine (75 mL) and stirred at0° C. Benzoyl chloride (1.2 mL, 10 mmol) was added, the reaction mixturewas stirred at room temperature for 15 h, then cooled to 0° C. Methanol(5 mL) was added, and the solvents were evaporated under reducedpressure. Ethyl acetate, Hexane and brine were added, and the organicextract was washed with brine, dried over sodium sulfate, filtered andevaporated to dryness. The residue was coevaporated with toluene anddissolved in a −15° C. solution of trifluoroacetic acid in water (90%v/v, 57 mL). The reaction mixture was stirred at −10° C. for 2 h, thenthe solvent was evaporated under high vacuum and low temperature. Theresidue was coevaporated with methanol, and a white precipitate wasformed. It was removed by filtration, and the filtrate containing thetitled compound was evaporated to dryness, and dissolved in ethylacetate, hexane and brine. The organic extract was washed with asaturated aqueous solution of sodium bicarbonate, washed with brine,dried over sodium sulfate, filtered, and the filtrate was evaporated todryness. The residue was subjected to silica gel chromatography [ethylacetate (33%) in hexanes] to give 1.8 g (63%, 3 steps) of the titledcompound as an oil.

Preparation of 1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-ethyl-β-D-ribofuranose

Benzoyl chloride (1.5 mL, 13 mmol) was added to a solution of5-O-benzoyl-4-C-ethyl-1-O-methyl-b-D-ribofuranose in pyridine (41 mL)stirred at 0° C., and the reaction mixture was stirred at roomtemperature for 18 h, then cooled to 0° C. Methanol (5 mL) was added,and the solvents were evaporated under reduced pressure. Ethyl acetate,Hexane and brine were added, and the organic extract was washed with a0.5 N solution of hydrochloric acid, then with brine, dried over sodiumsulfate, filtered and evaporated to dryness. The residue wascoevaporated with toluene and dissolved in a mixture of acetic acid (14mL) and acetic anhydride (1.5 mL). Sulfuric acid (97%, 165 mL) dilutedin acetic acid (1 mL) was added at 5° C. The reaction mixture wasstirred at 25° C. for 4 h and diluted with ethyl acetate (50 mL) andbrine (10 mL). The organic extract was washed with brine (3 times), witha saturated aqueous solution of sodium bicarbonate, washed with brine,dried over sodium sulfate, filtered, and the filtrate was evaporated todryness. The residue was subjected to silica gel chromatography [ethylacetate (0–3%) in dichloromethane] to give 2.9 g (92%) of the titledcompound as an oil (2:1 mixture of b/a anomers).

Preparation of 4-amino-5-oxo-pyrido[2,3-d]pyrimidine

Trimethylsilyl chloride (7.6 mL, 60 mmoles) was added to a stirredsuspension of 2-amino-3-cyano-4-methoxy-pyridine (Archiv der Pharmazie1985, 318, 481–486; 7.5 g, 50 mmoles) and sodium iodide (7.50 g, 50mmoles) in acetonitrile (225 mL). The resulting mixture was heated atreflux temperature for 24 h. The precipitate was filtered and washedwith ethyl acetate to give 10.4 g of a brownish powder(2-amino-3-cyano-4-oxo-pyridine). This powder was dried under reducedpressure, and suspended in 2-ethoxyethanol (300 mL). Formamidine acetate(31.2 g, 300 mmoles) was added, and the suspension was heated at refluxtemperature for 2 days, and then filtered. The grey residue obtained wasdissolved in a 2:1 boiling mixture of acetic acid and water, andcharcoal was added. The black suspension was filtered, and the filtratewas evaporated to dryness to give a white solid, which was suspended ina hot saturated solution of sodium hydrogencarbonate in water. Thesuspension was filtered to give 4-amino-5-oxo-pyrido[2,3-d]pyrimidine(2.0 g) as a white solid.

Preparation of4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine

4-Amino-5-oxo-pyrido[2,3-d]pyrimidine (0.23 g, 1.4 mmoles) was suspendedin 1,2-dichloroethane (20 mL) and the mixture was stirred at 55° C. BSA(0.87 mL, 3.5 mmoles) was added, the reaction mixture was stirred atreflux temperature for 90 min, then cooled to 40° C.1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-methyl-b-D-ribofuranose (0.60 g, 1.2mmoles) in 1,2-dichloroethane (3 mL) and TMSOTf (0.42 mL, 2.3 mmoles)were added to the clear solution, and the mixture was stirred at 100° C.for 48 h. The mixture was cooled to room temperature and a saturatedsolution of sodium hydrogencarbonate in water was added. The mixture wasdiluted with ethyl acetate (100 mL) and the organic extract was washedwith brine, dried over sodium sulfate, filtered, and the filtrate wasevaporated to dryness. The residue was subjected to silica gelchromatography [acetone (15–25%) in dichloromethane] to give 0.30 g(41%) of the titled compound as a white solid, and 0.14 g (19%) of4-amino-5-oxo-1-(2,3,5-tri-O-benzoyl-4-C-methyl-b-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid. Similarly, the following compounds were prepared:4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranosyl)pyrido-[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R,S)-C-ethynyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-5(R,S)-C-ethynyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-β-L-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-β-L-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R)-C-allyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-5(R)-C-allyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R)-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-5(R)-C-methyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-ethyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-4-C-ethyl-β-D-ribofuranose;4-Amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R,S)-C-vinyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidineas a white solid from 4-amino-5-oxo-pyrido[2,3-d]pyrimidine and1-O-acetyl-2,3,5-tri-O-benzoyl-5(R,S)-C-vinyl-β-D-ribofuranose.

Preparation of4-amino-5-oxo-8-(4-C-hydroxymethyl-β-D-ribofuranosyl)pyrido-[2,3d]pyrimidine

A solution of4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-benzoyloxymethyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine(0.82 g, 1.1 mmole) in methanolic ammonia (saturated at 0° C.) wasstirred in a sealed flask at 25° C. for 15 h. The solvents wereevaporated, and the residue was subjected to silica gel chromatography[methanol (30%) in dichloromethane] to give 0.36 g of the titledcompound as a white solid. Similarly, the following compounds wereprepared:4-Amino-5-oxo-8-(5(R)-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R)-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(5(R)-C-allyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R)-C-allyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(5(R,S)-C-ethynyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R,S)-C-ethynyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(5(R,S)-C-vinyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-5(R,S)-C-vinyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine from4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(β-L-ribofuranosyl)pyrido[2,3-d]pyrimidine from4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-β-L-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(4-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-methyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine;4-Amino-5-oxo-8-(4-C-ethyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidinefrom4-amino-5-oxo-8-(2,3,5-tri-O-benzoyl-4-C-ethyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.

Preparation of4-amino-5-oxo-8-(5(R,S)-C-ethyl-β-D-ribofuranosyl)pyrido-[2,3d]pyrimidine

Palladium on activated carbon (10% Pd, 200 mg) was added to a solutionof4-amino-5-oxo-8-(5-C-ethynyl-b-D-ribofuranosyl)pyrido[2,3-d]pyrimidine(0.14 g, 0.45 mmole) in methanol (50 mL). The flask was shaken under 3psi of hydrogen during 2 h and the catalyst was filtered and washed withmethanol. The solvents were evaporated, and the residue was subjected tosilica gel chromatography [methanol (10%) in dichloromethane] to give0.12 g of the titled compound as a white solid. Similarly, the followingcompounds were prepared:4-Amino-5-oxo-8-(5(R)-C-propyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine from4-amino-5-oxo-8-(5(R)-C-allyl-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.

Preparation of4-amino-5-oxo-8-(2-deoxy-β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine

A reaction mixture of4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine (1.8 g, 6.20mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (2.15 mL, 6.73mmol) in anhydrous pyridine (25 mL) was stirred at room temperature for20 h and cooled with ice. Water (0.5 mL) was added, and the mixturestirred at ambient temperature for 30 min and concentrated. The residuewas dissolved in ethyl acetate, washed with diluted sodium bicarbonate,dried over Na₂SO₄, and concentrated. Chromatography on silica(EtOAc-hexanes 3:2) gave 2.0 g of4-amino-5-oxo-8-[3,5-O-(1,1,3,3-tetraisopropyldisiloxy)-β-D-ribofuranosyl)]pyrido[2,3-d]pyrimidine.

To a solution of4-amino-5-oxo-8-[3,5-O-(1,1,3,3-tetraisopropyldisiloxy)-β-D-ribofuranosyl)]pyrido[2,3-d]pyrimidine(650 mg, 1.21 mmol) and DMAP (295 mg, 2.42 mmol) in acetonitrile (10 mL)was added phenyl chlorothioformate (185 mL, 1.33 mmol). The mixture wasstirred at room temperature for 2 h and concentrated to dryness. Theresidue was dissolved in chloroform, washed with water, dried (Na₂SO₄),and concentrated. The residue was dried under vacuum for 30 min and thendissolved in toluene (10 ML). 1,1′-Azobis(cyclohexanecarbonitrile) (74mg, 0.30 mmol) was added and the resulting solution was bubbled withargon for 30 min. Tris(trimethylsilyl)silane (0.56 mL, 1.82 mmol) wasadded and the resulting mixture stirred at 80° C. for 2 h and then at105° C. overnight. Solvent was evaporated and residue dissolved in THF(5 mL). TBAF (1.0 M in THF, 2.5 mL) was added and the resulting solutionstood at room temperature for 2 h and concentrated. Chromatography onsilica (10% MeOH in CH₂Cl₂) gave 240 mg of the titled compound.

In Vitro/In Vivo Experiments

Unless indicated otherwise, the following experiments were conductedwith 4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine.

Determination of EC₅₀

Cancer cells were incubated with4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine at variousconcentrations for a period of at least 24 hours and EC₅₀ wasdetermined. Table 1 gives in vitro activity of4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine in nMconcentrations.

Tumor Type Cell lines EC₅₀ [nM] Breast MCF 7, NCI/ADR-RES, MDA-MB-435,BT-549, 18 T-47D Prostate PC-3, DU-145 19 Kidney 786-0, A498, ACHN,CAKI-1, RXF 393, UO-31 44 Ovarian IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, 21SK-OV-3 Melanoma LOX IM VI, MALME-3M, M14, SK-MEL-2, 30 SK-MEL-28,ACC-257, UACC-62 CNS SF-268, SF-295, SF-539, SNB-19, SNB-75, U251 125Colon SW-620, KM 12, HT29, HCT-15, CHCT-116, 16 HCC-2998, COLO-205 LungNCI-H522, NCI-H460, NCI-H322M, NCI-H23, 68 HOP-92, HOP-62, EKVX, A549Leukemia SR, RPMI 8226, MOLT-4, K-562, HL-60, 34 CCRF-CEM LiverPLC/PRF5, Hep3B, Huh7 288 Pancreas PaCa-2, PANC-1 188

The relatively high efficacy of contemplated compounds, and especiallyof 4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine is furtherreflected in a series of experiments in which cytotoxicity andinhibition of proliferation of various cancer cell lines were comparedwith commercially available cytostatic agents. The results of theexperiments are shown in FIGS. 1 and 2.

Anti-Clonogenic Activity of Contemplated Compounds

To determine the anti-clonogenic activity of contemplated compounds invarious cancer cells experiments were conducted. The results of theseexperiments is shown in FIG. 3, clearly indicating that contemplatedcompounds exhibit significant anti-clonogenic activity, especially inmelanoma B16 and 140 cells, Leukemia cells (K562, M), and colon HT-29cells. Here again,4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine was used as arepresentative compound of contemplated compounds and compared againstEcyd and Gemzar.

Induction of Apoptosis

In order to determine how contemplated compounds interact with cancercells, NB4 cells (Leukemia) and Prostate 81 (prostate cancer cells)cells were incubated with4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine, Ecyd, andGemzar, and apoptosis was monitored by histone-DNA ELISA. The resultsare shown in FIGS. 4A and 4B and suggest that contemplated compoundsinduce apoptosis in a dose dependent manner.

Inhibition of RNA Synthesis

RNA synthesis was monitored in K562 cells using H³-Uridine incorporationfollowing a general protocol as outlined below. FIG. 5 shows inhibitionof RNA synthesis by exemplary contemplated compounds (here representedby 4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine) overvarious time periods and concentrations.

Inhibition of RNA Polymerase I, II, and III

To further investigate the influence of contemplated compounds (hereagain represented by4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine) on RNAsynthesis dependent on RNA Polymerase I, II, and III, experiments wereconducted. The results of these experiments are shown in FIGS. 6 and 7and suggest that contemplated compounds may inhibit processing of 28Sand 18S rRNA, however, fail to significantly inhibit RNA Polymerase III.The experiments further indicate that contemplated compounds inhibitRNA-Polymerase II dependent RNA synthesis in a concentration dependentmanner.

MEK-ERK Phosphorylation

It has recently been demonstrated [Wang, et al. JBC 275:39435–43 (2000);Pavlovic et al., Eur. Cytokine Netw 2:267–74 (2000)] that apoptosis canbe induced via the MEK-ERK signal transduction pathway. To investigatethe possibility of activation of the MEK-ERK signal transduction pathwayby contemplated compounds an experiment was conducted. FIG. 8 is anautoradiograph depicting phosphorylation of MEK and ERK, but not Raf bycontemplated compounds (represented by4-amino-5-oxo-8-(β-D-ribofuranosyl)pyrido[2,3-d]pyrimidine) in NB4leukemic cells. This, and other experiments suggest that thecontemplated compounds enhance phosphorylation of MEK (which is incontrast to Ecyd and Gemzar), however, they do not enhancephosphorylation of Raf. Consequently, it is contemplated that thecompounds according to the inventive subject matter may induce apoptosisthrough activation of MEK (which can be abolished by inhibitors of theMEK-ERK pathway).

Metabolites of Contemplated Compounds

Numerous experiments (data not shown) suggest that contemplatedcompounds are phosphorylated within a (tumor) cell, and that theproducts include mono-, di-, and triphosphorylated forms (e.g.,detectable by LC-MS). It is further contemplated that the metabolitesmay have significant (or even increased) biological activity.

Thus, specific embodiments and applications of pyrido[2,3-d]pyrimidineand pyrimido[4,5-d]pyrimidine nucleosides have been disclosed. It shouldbe apparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced.

1. A nucleoside analog according to the formula (I)

wherein A is O; R₂, R₂′, R₃, and R₃′ are independently selected from H,F, OH, NH₂, CN, N₃, CONH₂, and R, where R is lower alkyl, lower alkenyl,lower alkynyl, or lower acyl; or R₂ and R₂′ together, or R₃ and R₃′together are selected from ═CH₂, ═CHR″, ═CR″₂, ═NR″, where R″ is H, F,OH, CN, N₃, CONH₂, lower alkyl, lower alkenyl, lower alkynyl, or loweracyl; R₄ is selected from H, lower alkyl, lower alkenyl, lower alkynyl,aralkyl, or hydroxymethyl; R₅′ is selected from H, lower alkyl, loweralkenyl, lower alkynyl, or aralkyl; R₅ is H, OH, OP(O)(OH)₂, orP(O)(OH)₂; and B is selected from the group of heterocyclic radicalsconsisting of formulae (II) and (IV)

wherein X is H, NH₂ or OH; Y is H, NH₂, or halogen; Z₁ is O, S, NR″,CHM, or CM₂; Z₃ is N, CH, or CM; Z₂ is N, CH, or CM; where M is F, Cl,Br, OH, SH, NH₂, CN, COOR″, C(═NH)NH₂, lower alkyl, lower alkenyl, loweralkynyl, aralkyl, or aryl.
 2. The nucleoside analog of claim 1 wherein Ais O, and B is a heterocyclic radical according to formula (II).
 3. Thenucleoside analog of claim 2 wherein X is NH₂, Z₁ is O, and Z₂ and Z₃are CH.
 4. The nucleoside analog of claim 3 wherein R₄ and R₅′ arehydrogen, and R₅ is OH.
 5. The nucleoside analog of claim 1 having astructure according to formula (VI)


6. A prodrug comprising the nucleoside analog of claim
 5. 7. The prodrugof claim 6 wherein the prodrug comprises a phosphate or phosphonatecovalently coupled to the C₅ atom of the ribose.
 8. The prodrug of claim6 wherein the prodrug comprises a moiety that is covalently bound to atleast one of the hydroxyl groups of the ribose, and that is cleaved fromthe at least one hydroxyl groups within a target cell.
 9. The prodrug ofclaim 6 wherein the prodrug comprises a moiety that is covalently boundto the amino group of the base, and that is cleaved from the amino groupwithin a target cell.
 10. A method of inhibiting growth of a neoplasticcell comprising: providing a compound according to claim 1; andpresenting the compound to the cell in a dosage effective to inhibit thegrowth of a cell.
 11. The method of claim 10 wherein A is O, and B is aheterocyclic radical according to formula (II).
 12. The method of claim11 wherein X is NH₂, Z₁ is O, and Z₂ and Z₃ are CH.
 13. The method ofclaim 12 wherein R₄ and R₅′ are hydrogen, and R₅ is OH.
 14. The methodof claim 10 wherein the compound has a structure according to formula(VI)


15. The method of claim 14 wherein the compound comprises a phosphate orphosphonate covalently coupled to the C₅ atom of the ribose.
 16. Themethod of claim 14 wherein the compound comprises a moiety that iscovalently bound to at least one of the hydroxyl groups of the ribose,and that is cleaved from the at least one hydroxyl groups within atarget cell.
 17. The method of claim 14 wherein the compound comprises amoiety that is covalently bound to the amino group of the base, and thatis cleaved from the amino group within a target cell.
 18. The use ofclaim 14 wherein the neoplastic cell is a cell selected from the groupconsisting of a colon cancer cell, a breast cancer cell, a melanomacell, a glioma cell, prostate cancer cell, a lung cancer cell, a livercancer cell, a pancreas cancer cell, and an ovarian cancer cell.
 19. Themethod of claim 14 wherein the inhibition of the growth of the cellcomprises apoptosis.
 20. The method of claim 19 wherein the apoptosis istriggered at least in part by MEK-phosphorylation.
 21. The method ofclaim 14 wherein the inhibition of the growth of the cell comprisesinhibition of at least one of RNA polymerase I, RNA polymerase II, andRNA polymerase III.